Others have reported that unfolding free energy is only 2.1 and 3.1 kcal per mole, respectively for yeast iso-1-cytochrome c containing Cys 102 blocked with iodoacetamide and wt iso-2 while it is 7.3 kcal per mole for horse cytochrome c. Despite recent advancement in understanding of thermostability of proteins, the origin of this marked stability difference is not clear. To assess the origin we considered the following observations: (i) Others have shown that the three-dimensional structures of yeast iso-2- and iso-1-cytochromes c are similar to horse cytochrome c. (ii) We substituted the hydrophobic core residues of iso-2 with those corresponding to horse cytochrome c. This chimera was further mutated such that all Gly and Pro residues of the corresponding amino acid sequence correspond to those of horse cytochrome c. The resulting mutant, called the horse core-iso-2 shell chimera, showed distinctly lower stability than would be expected if it were similar to horse cytochrome c (iii) The calorimetric data of others indicate that the stability difference between horse cytochrome c and yeast iso-2 cytochrome c has an entropic origin. (iv) The number of hydrogen bonds identified by others is greater for iso-1 or iso-2 than for horse cytochrome c. (v) The net formal charges (excluding His) are +7, +5 and +7, respectively for horse cytochrome c, iso-1 and iso-2. (vi) Our previous data of fragment complexes of cytochromes c suggest that a distinct decrease in free energy of horse cytochrome c relative to iso-1 may occur in the late phase of folding, where a majority of the hydrophobic core is formed. (vii) Analysis of our previous data has led to the idea that an interaction between the hydrophobic core and surface residues would also be significant in the late phase of folding. (viii) Our previous data of truncated fragment complexes of cytochrome c suggests that at least those four N-terminal of the five extra N-terminal residues of iso-1, as compared with horse cytchrome c, probably would not govern the stability difference. (ix) Data of others suggest that the stability difference in question would be reflected in the difference in the energy barrier of unfolding but not folding. (x) The report of others also suggests that there may be interactions between the interior residues and positive charges on the surface of horse cytochrome c, which would stabilize the protein and that such interactions do not exist in the molten globule state. The question to be addressed in the present study is the nature of the interaction between hydrophobic core and surface residues. To this end we investigated the following system. Others have reported that phosphate stabilizes horse cytochrome c. There are three phosphate-binding sites reported by others for horse cytochrome c that are located on the surface. Others also suggested that the major force of phosphate binding would be interactions with positive charges. Thus, investigation whether mutation of the hydrophobic core residues would perturb the phosphate binding would serve as a probe whether the hydrophobic core could interact with the surface charges. We investigated the influence of hydrophobic core residues on phosphate binding by mutating residues in yeast iso-2-cytochrome c to those corresponding to iso-1-cytochrome c in various combinations. Heat transition of ultraviolet CD was followed as a function of pH in the presence and absence of phosphate. Thermodynamic parameters were deduced. It was found that the I20V/V43A/M98L mutation in the hydrophobic core, whose locations are remote from the putative phosphate sites, modulates phosphate interactions. The modulation is pH dependent. The I20V/M98L and V43A mutation effects are non-additive. The results lead to a model analogous to that of Tsao-Evans-Wennerstrom where a domain associated with the ordered hydrophobic core is sensitive to the fields generated by the surface charges. Such an explanation would be in accord with the observed difference in thermal stability between iso-2 and horse cytochromes c. The present model of a polarizable domain associated with the hydrophobic core of cytochrome c gives an explanation of the cooperative interaction i.e. many-body force of the second origin that is distinct from hydrophobic interactions. We have hypothesized that such a cooperative interaction of the second origin would be important for stabilizing proteins in the late phase of folding.